PROJECT SUMMARY The goal of this research is to identify direct substrates of the Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. These kinases are essential both for M. tuberculosis viability in vitro and during infection. Together with the success of kinase inhibitors as clinically useful drugs in other settings, their essentiality makes PknA and PknB potentially valuable targets for new antituberculosis drugs. A large number of phosphoproteins have been identified in M. tuberculosis; for relatively few of these, however, is the cognate kinase that phosphorylates the protein known. This research aims to address this knowledge gap. This research proposal has two Aims. The first aim makes use of modified kinase proteins together with ATP analogs that are specifically utilized by the modified kinases. These reagents are used to specifically label proteins that are substrates of either PknA or PknB. Labeled proteins can then be covalently captured, purified and identified by mass spectrometry. Proteins identified in this process that are not present in control assays are high probability direct substrates of the kinase of interest. The second aim makes use of strains in which the kinase of interest, PknA, PknB or both PknA and PknB are depleted. The total phosphoproteomes of these kinase-depleted strains, as well as control strains expressing native levels of the kinases, are then analyzed using optimized methods for phosphoprotein enrichment and mass spectrometry-based quantification. Phosphopeptides absent or significantly decreased in the kinase- depleted strain compared to the control strain are candidate substrates of the kinase that is depleted. Combining the results of the two aims of this research will identify substrates of PknA and PknB. These data will provide new insights into regulatory functions of these kinases and provide the basis to investigate the mechanisms by which phosphorylation of substrate proteins affects their function in the M. tuberculosis cell.